Glycosylation is the process or result of addition of saccharides to proteins and lipids. It is a common co-translational or post-translational modification of proteins. The majority of proteins synthesized in the rough endoplasmic reticulum undergo glycosylation. It is estimated that more than half of all cellular and secretory proteins are glycosylated (Apweiler et al., 1999, Biochim. Biophys. Acta 1473: 4-8). Glycosylation is important for modulating normal cellular processes and recognition events. Changes in the carbohydrate moieties of the glycan also may change normal glycoprotein function. Aberrations, such as the changes in the amount of fucose, sialic acid, glucose, galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, a Lewis antigen, or N-linked β(1,6)-branching in the glycan portion of the core glycan, have been observed in several disease processes, including cancer. In mammals, over-expression of fucosyltransferases leading to increased fucosylation of cell surface glycoproteins has been correlated with malignancy and increased metastatic potential in breast, liver, lung, and ovarian cancer. Because the tumor-associated fucosylated or Lewis antigen containing proteins are often liberated from the cell surface and can be detected in the blood stream (Orntoft and Vestergaard, 1999, Electrophoresis 20: 362-371), pathological changes are likely to be reflected in serum protein profiles. While previous studies have reported increased fucosylation or sialylation of Lewis antigens on specific proteins isolated from serum of cancer patients, these single proteins are often non-specific and are present in other types of disease or neoplasia.
Presently, diseases are largely diagnosed, and patient status is monitored, through the use of clinical chemistry analyses and/or analyses of individual proteins. By and large, these are not as specific or as sensitive as is desirable. For example, while serum concentrations of prostate specific antigen (PSA) are used for the detection of prostate cancer and for monitoring its recurrence following treatment, this technique is not very sensitive, has a high rate of false positives, and the PSA serum concentrations do not correlate with the seriousness of the cancer. Similarly, the serum activity of alanine aminotransferase (ALT) is used to detect and monitor liver damage. While widely used, usefulness of this approach is limited because an increase in serum ALT activity does not give an indication of what is causing the liver damage, and significant damage to the liver can occur before the increase in serum ALT activity, thus ALT activity cannot always be used reliably to monitor liver damage.
Protein biomarker profiling holds the promise of enabling increased diagnostic and prognostic monitoring of disease, treatment efficacy, and general health. Current methods to detect the presence of protein biomarkers associated with a specific disease state include separating proteins by their mass (SDS-PAGE), charge (isoelectric focusing and 2-D gel electrophoresis) and immunoreactivity (ELISA). While these techniques allow qualitative or quantitative detection of a particular biomarker, use of these methods in discovering novel biomarkers is hindered by insufficient sensitivity, specificity, lack of quantitative measurement, and/or they are time intensive. Finally, many types of cancers and other diseases currently have no clinically useful biomarkers.
Mass spectrometry-based strategies for protein identification and quantification have made it possible to perform global, large scale comparative proteomics in complex biological samples. However, current methods in proteomics are generally inadequate for the study of glycoproteins. Two-dimensional gel electrophoresis is not rapid enough for routine diagnostic use. Multidimensional chromatographic methods suffer in that changes in glycosylation have little impact on retention time in reversed phase chromatography. Of importance for diagnostics, existing methods for quantifying significant disease-related differences in glycosylation are unsatisfactory, particularly at a high throughput proteomics level.
The merits of targeting glycans as a way to identify proteins might seem unusual. The logic behind this strategy stems from the fact that in the case of glycoproteins, glycan structure often changes in association with disease (Yamada et al., 2003, Oncol. Rep. 10: 1919-1924). The fact that these aberrant glycans can also be immunogenic has been exploited by pathologists in detecting cancer. Through the use of fluorescently labeled glycan-directed antibodies, staining procedures have been developed that allow differentiation between normal and malignant cells in tissue on the basis of targeting aberrant glycosylation (Edwards et al., 1986, Cancer Res. 46: 1306-1317). In addition, surface glycoproteins are well documented to play a prominent role in the loss of cellular adhesion, metastasis, the binding of tumor cells at remote sites, and secondary tumor colonization. For example, the Lewis (Le) antigens a (Lea), b (Leb), x (Lex), and y (Ley), and their sialylated forms s-Lea, s-Leb, s-Lex, and s-Ley, are among the more important glycans involved in these processes (Brockhausen, 2006, EMBO Rep. 7: 599-604). Cancer-associated glycoproteins carrying these glycans have been reported to be shed into blood and lymph as well (An et al., 2006, J. Proteome Res. 5: 1626-1635). Unfortunately, the proteins associated with cancer-associated glycans are generally unknown. If suitable analytical methods were developed and the structures of the glycoproteins were determined, they could potentially be used as cancer biomarkers.
The value of affinity selection in glycoprotein identification has been well established with lectins (Drake et al., 2006, Mol. Cell. Proteomics 5: 1957-1967). Immobilized lectins that seek out disease-related features of glycans reduce the complexity of blood samples sufficiently so that glycoproteins can be identified by shotgun proteomics without abundant protein removal (Rosenfeld et al., 2007, J. Biochem. Biophys. Methods 70: 415-426). Glycoproteins have also been identified through affinity selection of their glycopeptides from tryptic digests (Xiong et al., 2003, J. Proteome Res. 2: 618-625). After deglycosylation of the affinity selected glycopeptides are further fractionation by RPC, peptides carrying the glycan binding site can be identified by tandem mass spectrometry (Qiu and Regnier, 2005, Anal. Chem. 77: 7225-7231).
Present testing for cancer and other diseases relies on testing serum for the concentrations of single proteins which are loosely correlated to the disease, or for the activities of serum enzymes which are generally indicative of abnormal function of one or more organs. Increases in both the specificity and sensitivity over these current tests would greatly improve the accuracy and timeliness of the diagnosis and allow improved treatment selection and patient monitoring. New, more sensitive and specific markers of diseases, in particular cancers, are needed. The invention described here addresses these and related needs.